Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.     

Activation and proteasomal degradation of rho GTPases by cytotoxic necrotizing factor-1 elicit a controlled inflammatory response

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3alpha (MIP-3alpha) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3alpha, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses.     

Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains

Cytotoxic necrotizing factor type 1 (CNF1) induces, in epithelial cells, the development of stress fibres via the GTPase Rho pathway. We showed that CNF1 is able to modify Rho both in vitro and in vivo. Recombinant N-terminal 33 kDa (CNF1Nter) and C-terminal 14.8–31.5 kDa (CNF1Cter) regions of the CNF1 protein allowed us to demonstrate that the N-terminal region contains the cell-binding domain of the toxin and that the C-terminal region is responsible for its catalytic activity. CNF1Nter lowered the activity of CNF1 when provided to cells before the toxin whereas CNF1Cter had no effect on CNF1 cell toxicity. CNF1Cter was sufficient to induce a typical CNF1 phenotype when microinjected into African green monkey kidney cells (Vero cells), and was able to modify Rho as previously reported for CNF1. The C-terminal domain lost its catalytic activity when deleted of various subdomains, suggesting a scattered distribution of catalytic-site amino acids. Elucidation of the CNF1 functional organization and analysis of amino acid homologies between CNFs (CNF1, CNF2), Pasteurella multocida toxin (PMT) and dermonecrotic toxin of Bordetella pertussis (DNT) allowed us to postulate that CNFs and DNT act on Rho via the same enzymatic activity located in their C-terminus, and that CNFs and PMT probably bind to analogous cell receptors.     

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.     

CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.     

37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.     

Cytotoxic necrotizing factor 1 prevents apoptosis via the Akt/IkappaB kinase pathway: role of nuclear factor-kappaB and Bcl-2

Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-B-induced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/IkappaB kinase/nuclear factor-kappaB prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed.     

CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells

Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.     

Identification of the region of rho involved in substrate recognition by Escherichia coli cytotoxic necrotizing factor 1 (CNF1).

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.     

Cytotoxic necrotizing factor 1 fromEscherichia coli: a toxin with a new intracellular activity for eukaryotic cells

Certain pathogenicEscherichia coli strains elaborate a toxin, the cytotoxic necrotizing factor type 1 (CNF1). CNF1 covalently and specifically modifies the p21 Rho GTP-binding protein in mammalian cells by deamidation of the p21 Rho glutamine 63. CNF1 modification of Rho leads to permanent activation of the GTP-binding protein by blocking intrinsic and RhoGAP GTPase activities. Rho activation by CNF1 induces reorganization of the actin cytoskeleton into large stress fibers and the multiplication of focal contact points. Deamidation is a new catalytic activity described for an intracellularly acting toxin. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Rho GTPases as targets of bacterial protein toxins

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.     

The Rac-activating toxin cytotoxic necrotizing factor 1 oversees NK cell-mediated activity by regulating the actin/microtubule interplay

The cell cytoskeleton is widely acknowledged as a master for NK cell function. Specifically, actin filaments guide the NK cell binding to target cells, engendering the formation of the so-called immunological synapse, while microtubules direct the killer behavior. All these cytoskeleton-dependent activities are competently governed by the Rho GTPases, a family of regulatory molecules encompassing the three different subfamilies, Rho, Rac, and Cdc42. By using a Rac GTPase-activating bacterial protein toxin from Escherichia coli named cytotoxic necrotizing factor 1 (CNF1), we obtained results supporting the activation of Rac GTPase as a booster for effector cell-binding efficiency, recruitment ability, and, consequently, cytotoxicity. In particular, the augmented killer capacity of CNF1-treated NK cells was associated with the increased expression of certain cell adhesion or activation-associated molecules and the reshaping of the actin and microtubule networks. Importantly, CNF1 counteracted the activity exerted by toxins disrupting the cytoskeletal architecture. Hence, the activation of Rho GTPases, particularly Rac, induced by CNF1, appears to orchestrate a dynamic cross talk between microtubules and actin filaments, leading to a fruitful NK cell activity and polarization state. Our findings suggest that protein toxins might be viewed as modulators of NK cell cytotoxic activity and could possibly be regarded as useful pharmacological tools for certain Rho-linked immune diseases in the near future.     

Isotype-specific degradation of Rac activated by the cytotoxic necrotizing factor 1

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.     

Change in substrate specificity of cytotoxic necrotizing factor unmasks proteasome-independent down-regulation of constitutively active RhoA

Cytotoxic necrotizing factors CNF1 and CNF2 are produced by pathogenic Escherichia coli strains. They constitutively activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. Recently, a novel CNF (CNF(Y)) encompassing 65% identity to CNF1 has been identified in Yersinia pseudotuberculosis. In contrast to the E. coli toxins, which activate several isoforms of Rho family GTPases, CNF(Y) is a strong and selective activator of RhoA in vivo. By constructing chimeras between CNF1 and CNF(Y), we show that this substrate specificity is based on differences in the catalytic domains, whereas the receptor binding and translocation domains have no influence. We further define a loop element (L8) on the surface of the catalytic domains as important for substrate recognition. A single amino acid exchange in L8 is sufficient to shift substrate specificity of CNF1. Moreover, it is shown that RhoA activation by CNF1 is transient, which may be the consequence of the broader substrate specificity of the E. coli toxin, leading to cross-talk between the activated GTPases.     

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

Deamidation of RhoA glutamine 63 by the Escherichia coli CNF1 toxin requires a short sequence of the GTPase switch 2 domain

CNF1, a toxin produced by pathogenic Escherichia coli strains, deamidates the RhoA GTP-binding protein glutamine 63 and impairs RhoGAP-mediated GTP hydrolysis resulting in RhoA permanent activation. Using peptides derived from the RhoA sequence, we found that DTAGQEDYDRL (corresponding to RhoA 59-69 residues) was the minimum RhoA-derived peptide which could be deamidated in vitro by the CNF1 catalytic domain (CNF1-Cter). Site-directed mutagenesis outside the RhoA 59-69 sequence had no influence on glutamine 63 deamidation by CNF1-Cter. RhoA proteins with substitutions L57G, D65G, Y66G, or R70G were not affected in their ability to be deamidated by CNF1-Cter, whereas this was abolished by the R68G substitution. Arginine 68 is part of the DYDRL motif that is strictly conserved in Rho, Rac, and Cdc42 but not in other small GTP-binding proteins consistent with the observation that only Rho, Rac, and Cdc42 can be modified by CNF1.     

Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.     

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.